ObjectiveThe gray-zone prostate-specific antigen (PSA) range is accepted to be 4-10 ng/ml and is considered to vary according to age. We aimed to investigate whether fusion prostate biopsy (FPB), which has been reported to have relatively higher cancer detection rates, has an effect on gray-zone PSA cut-off value.Material and methodsThis retrospective study included patients that underwent standard prostate biopsy (SPB) or multiparametric magnetic resonance imaging (MpMRI)-guided FPB (SPB+ targeted biopsy). All the patients included in the study were detected with a Prostate Imaging Reporting and Data System (PI-RADS) ≥3 lesion on MpMRI (the FPB group only). The demographics, clinical characteristics, and histopathological diagnoses were recorded for each patient.ResultsA total of 1,628 patients comprising 1,208 patients in the SPB group and 420 patients in the FPB group were included in the study. The mean PSA level was 9.75±6.68 ng/ml in the FBP group and 10.46±6.46 ng/ml in the SPB group (p=0.053). Prostate cancer (PCa) detection rate was significantly higher in the FPB group as compared to the SPB group (42.4% vs. 36.4%). The PSA cut-off value for PCa was 9.75 ng/ml  in the SPB group and was 7.55 ng/ml  in the FPB group. In the FPB group, the cancer detection rate among the patients with a PSA level of 7.55-10.00 ng/ml was 56.1%.ConclusionThe results indicated that the introduction of FPB into clinical practice, which has relatively higher cancer detection rates, has further lowered the upper limit for gray-zone PSA. Prostate cancer (PCa) is the second-most common cancer in men worldwide . ProstatThere is no consensus in the literature regarding the normal PSA range. However, the upper limit for PSA is often accepted to be 2.5 or 4.0 ng/ml, and it is considered to vary according to age -9.Multiparametric magnetic resonance imaging (MpMRI)-guided fusion prostate biopsy (FPB) is a recently developed biopsy technique. This technique has been shown to provide effective outcomes. FPB can be performed in the form of cognitive, fusion, or in-bore biopsy using ultrasound fusion imaging with a computer program . NumerouThe present study aimed to investigate whether FPB, which has relatively higher cancer detection rates, has an effect on gray-zone PSA cut-off value by comparing the PSA cut-off values and gray-zone PSA ranges for SPB and FPB.PatientsThe retrospective study included patients aged 45-75 years that underwent either 10-12 core SPB between January 2010 and December 2016 or mpMRI-guided FPB (12 core SPB + targeted biopsy (TB)) between January 2017 and April 2019. Inclusion criteria were elevated PSA levels and/or the presence of suspected rectal examination-suspicious radiological imaging. Patients with a Prostate Imaging Reporting and Data System (PI-RADS) <3 lesion, PSA >50 ng/ml, incomplete medical records, a histopathological diagnosis of atypical small acinar proliferation (ASAP), or high-grade prostatic intraepithelial neoplasia (HGPIN), and patients that underwent SPB with fewer than 10 cores or more than 12 cores were excluded from the study. Demographic and clinical characteristics, including age, body mass index (BMI), serum PSA level, total prostate volume, number of biopsy cores, and histopathological diagnosis, were recorded for each patient.Multiparametric MRI and Prostate Imaging-Reporting and Data SystemPrior to FPB, each patient underwent prostate mpMRI without endorectal coil using a Siemens Magnetom 1.5 Tesla MRI device . Suspicious lesions detected on contrast-enhanced T1-, T2-, and diffusion-weighted sequences were recorded based on PI-RADS version 2 . In patiPre-biopsyPrior to biopsy, each patient gave a sterile urine culture. The ongoing anticoagulant and antiaggregant treatments of the patients were stopped. Twenty-four hours before the procedure, three oral doses of 750 mg of ciprofloxacin were administered at a 12-h interval. No bowel preparation was performed in any patient prior to the procedure. Biopsy procedures were performed under local or sedation anesthesia in the outpatient clinics according to patient preference.Standard prostate biopsySPB was performed under transrectal ultrasonography (TRUS) guidance with 10-12 cores. Local anesthetics were administered, followed by the injection of intrarectal lignocaine gel + prilocaine combination. Following the induction of periprostatic nerve blockage, total prostate volume was measured using the following formula: height (H) x width (W) x length (L) x 0.523.Fusion prostate biopsyFPB was performed by using an ultrasonography (US) system with rigid fusion software and an endorectal single-angle probe. Local anesthetics were administered, followed by the injection of intrarectal lignocaine gel + prilocaine combination. The presence of prominent lesions was determined by a sonographic examination of the prostate tissue. Total prostate volume was measured using the following formula: H x W x L x 0.523. FPB was performed with 12 cores and then the mpMRI images were transferred to the US system. Segmentation (matching) of sonographic images with MR images was performed and the lesions detected on mpMRI were marked. For each lesion marked on mpMRI, FPB (SPB + TB) was performed using two to five cores for each lesion. All the data transfers and markings throughout the procedure were performed by two urologists experienced and trained in transrectal prostate ultrasonography and biopsy.Histopathological examinationThe specimens were sent for a histopathological examination in previously labeled containers. The cancer detection rate per core was calculated in accordance with the International Society of Urological Pathology (ISUP) grading system, which is based on primary and secondary Gleason scores . ClinicaStatistical analysisData were analyzed using Statistical Package for Social Sciences (SPSS) for Windows version 22.0 . Normal distribution of data was assessed with the Shapiro-Wilk test and histogram plots. Based on the distribution pattern, continuous variables were presented as mean ± standard deviation (SD) or as median (1-3rd quartile). Categorical variables were expressed as percentages (%). In independent groups, normally distributed continuous variables were compared using independent-samples t-test and non-normally distributed continuous variables were compared using the Mann-Whitney U test. The cut-off value was considered as the point with the maximum sensitivity and specificity and was determined using a Receiver Operating Characteristic (ROC) curve analysis. A p-value of <0.05 was considered significant.A total of 1,628 patients comprising 1,208 (74.2%) patients in the SPB group and 420 (25.8%) patients in the FPB group were included in the study. The mean PSA level was 10.28 ± 6.52 ng/ml. Of all the patients, 38% of them were detected with sPCa, among whom ISUP grade 1 was the most common histopathological diagnosis (17.8%). Table The two groups were similar with regard to PSA level and median age . Overall PCa and sPCa detection rates were significantly higher in the FPB group as compared to the SPB group Table .In the ROC curve analysis, the area under the ROC curve (AUC) for PSA was 0.837 in the FPB group and 0.849 in the SPB group and a strong correlation was found between PCa and PSA in both groups Figure . The PSAIn the SPB group, the PSA cut-off value for PCa was 9.75 ng/ml, which appears consistent with the commonly accepted gray-zone PSA level (10 ng/ml). In the FPB group, however, the PSA cut-off value for PCa was 7.55 ng/ml, which supports the hypothesis that the upper limit for gray-zone PSA has been further lowered with the introduction of FPB into clinical practice. On the other hand, the cancer detection rate in the FPB group among the patients with a PSA of 7.55-10.00 ng/ml was 56.1%, which implicates that this reference range cannot be accepted as the gray-zone range.In Europe, prostate cancer is the second most common cancer in men and age-adjusted prostate cancer incidence rate was 35 cases per 100,000 in Turkey . The mosFPB is known to detect more prostate cancer than SPB at low PSA levels -13. AccoBoasen et al. , in theiIn our patients, ISUP grade 1 was the most common histopathological diagnosis (46.8%). Similarly, a 2013 study that evaluated 582 patients also reported that ISUP grade 1 was the most common histopathological diagnosis in the patients . A more Our study has several key limitations. First, the study had a retrospective design. Second, a large patient series is needed for the determination of a novel and definitive cut-off value for gray-zone PSA. Therefore, the relatively small number of patients in the FPB group is an important limitation of our study. Third, the presence of a well-established cancer marker (i.e. PI-RADS ≥3 lesion) in our patients that underwent FPB may lead to partial bias. This could be accepted as a limitation of our study; however, it should be recognized that FPB and the presence of a PI-RADS ≥3 lesions are highly integrated with each other. Fourth, patients that underwent FPB and had a histopathological diagnosis of ASAP and HGPIN were excluded from the study and no clear information could be obtained regarding their treatment outcomes due to their short follow-up period, which might have led to the acquisition of results that could be slightly different from those of daily practice. Finally, an age-based PSA cut-off value could not be calculated as there were very few patients in some age groups.The results indicated that the introduction of FPB into clinical practice, which has relatively higher cancer detection rates, has further lowered the upper limit for gray-zone PSA. Accordingly, we suggest that the upper limit for gray-zone PSA should be revised to 7.5 ng/ml and the clinics administering FPB should be more courageous in making biopsy decisions in patients with a PSA level of >7.5 ng/ml.


Dictyophora indusiata (Vent. Ex. Pers.) Fischer or Phallus indusiatus is an edible member of the higher mushroom phylum of Basidiomycetes. Known for its morphological elegance that gave it the names bridal veil fungus, veiled lady or queen of the mushrooms, it has numerous medicinal values that are beginning to be acknowledged through pharmacological efficacy studies. In an attempt to promote research on this valuable natural resource, the present communication aims to provide a comprehensive review of the chemistry, pharmacology and potential therapeutic applications of extracts and compounds isolated from D. indusiata. Of the bioactive compounds, the chemistry of the polysaccharides as major bioactive components primarily the β-(1→3)-D-glucan with side branches of β-(1→6)-glucosyl units are discussed, while small molecular weight compounds include terpenoids and alkaloids. Biochemical and cellular mechanisms of action from general antioxidant and anti-inflammatory to more specific signaling mechanisms are outlined along with potential applications in cancer and immunotherapy, neurodegenerative and chronic inflammatory diseases, etc. Further research areas and limitations of the current scientific data are also highlighted. Dictyophora indusiata (Vent.) Fisch. is an edible and medicinal mushroom that belongs to the family Phallaceae of the Agaricomycetes class (phylum Basidiomycetes) of fungi. In recent taxonomic literature, the synonym Phallus indusiata Vent. is the accepted name for the fungus though nearly all the scientific literature so far is available under the name entry of D. indusiata. As a saprophytic fungus, it grows in well-rotted woody trunk or rich soil of tropical Africa, Asia, Australia and the Americas. Its food and medicinal value are however much appreciated in the far eastern countries such as China where it grows on the wet roots of bamboo groves and in forests. Its common local names mainly in China and Japan include bamboo mushrooms, bamboo pith, long net stinkhorn, crinoline and stinkhorn basket, but perhaps the names most vividly associated with the morphologically distinctive feature of the fungus are bridal veil fungus, veiled lady or queen of the mushrooms. As shown in D. indusiata has three macroscopic features: conical cap, a stalk and an indusium net-like white veil that hangs from the head/cap down to cover the leg/stalk. Hence, the name-veiled lady appears to be given to describe the skirt like appearance of the elegant fruiting body -D-glucan with side branches of β-(1→6)-glucosyl units. The molecular mass and total sugar content were 536 kDa and 97.6% respectively. Various molecular weight polysaccharides have been identified however and a range of 801–4656 kDa for six polysaccharides have been described [D. indusiata is now well established as a (1→6)-branched, (1→3)-β-d-glucan and is as depicted in α-D-mannan, branched (1→3)-β-D-glucan and the location of the O-acetylation of the (1→3)-α-D-mannan [Wang et al.  establiseneous β-→3-D-glucs of β-1→-glucosylD-mannan ,11,12. PD-mannan  in theirβ-glucans have also been developed of which the fluorometric assay based on aniline blue dye described by Fu et al. [β-glucans as 1.20% and total polysaccharides as 5.41%. Wu et al. [D. indusiata. For the three kinds of enzymes  employed, they established an optimum extraction temperature of 52.5 °C, extraction time 105 min and pH of 5.25. Considering the predicted polysaccharide yield of 9.87%, the enzyme-assisted extraction yield of 9.77 ± 0.18% appear to be an exhaustive extraction method. On the other hand, Liu et al. [D.indusiata. Under the optimum extraction time of 2.1 h, solid to liquid ratio of 1:37, and extraction temperature of 92 °C, they were able to enhance the yield to 15.95%. Water-soluble polysaccharides have also been routinely extracted by ultrasonic extraction .D.indusiata polysaccharides are normally prepared by water extraction followed by centrifugation, removal of proteins and alcohol precipitation. As a general approach, the dried fruiting body may be subjected to organic solvent extraction  to remove lipophilic components and the remaining part can be extracted by either of the methodologies mentioned above. In many cases, hot or boiling water extraction by distilled/deionized water for up to 2 h are performed for initial extraction. After filtration and removal of water (rotary evaporator or freeze drying), proteins are removed -β-glucan side chain residues had the molecular weight of 650 kDa, and the total sugar and uronic acid contents were 95.67% and 0.63% respectively [β-glucan polysaccharide from D. indusiata [ method, ). The crectively . A previndusiata . Anion-endusiata  to isolaD. indusiata. As discussed in the following sections, the activity profile of the crude polysaccharides could be different from the isolated one single polysaccharide. The acid and alkali extracts do also have different compositions with glucose predominant in both cases but Glc, Fru and Man for the acid and Glc and Fru for the alkali extract [As with small molecular weight drug molecules, the effect of polysaccharides should not be taken as one common pharmacological property. Individual active components must therefore be isolated if one needs to assess the true potential of all polysaccharides in  extract . Further extract  establis extract .D. indusiata polysaccharides is predominantly of the β-(1→3)-D-glucan with side branches of β-(1→6)-glucosyl units that could vary depending on the composition of monosaccharides and their molecular weight. The (1→3)-α-D-mannopyranosyl residues that contain O-acetyl group do, however, also occur.Overall, the structural feature of D.indusiata was established by the work of Ishiyama et al. [3R, 4S)-3, 7-dimethyl-1, 6-octadien-3, 4-diol which was also found as an oleoyl or linoleoyl ester derivative at the 3-hydroxyl position -2-furfural formation from sugars is elegantly presented by Antal et al. [Sharma et al.  identifil et al. .D. indusiata is shown in An overview of in vitro and in vivo effects of extracts and compounds isolated from ·) was generated through iron catalyzed H2O2 fission, the water-soluble polysaccharides have been shown to display free radical scavenging effect [· and superoxide (O2−) radicals have also been shown [+ and OH·, DPPH, and O2− radicals were suppressed wile lipid peroxidation was inhibited both by the water extract, crude polysaccharides and their sub-fractions [2− and DPPH radicals when tested at the concentration range of 0.2–1.4 mg/mL [β-d-glucan polysaccharide was further shown [In an experiment where hydroxyl radical  has been shown to demonstrate antioxidant and anti-hemolytic effect at exceptionally low dose of 20 nmol/mL. While the level of lipid peroxidation (LPO) marker, malondialdehyde (MDA), and reactive oxygen species (ROS) were suppressed, the activities of intracellular antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) were enhanced. Furthermore, the cupric chloride-induced conjugated diene formation in plasma was ameliorated by the DP1 ).Caenorhabditis elegans, D. indusiata polysaccharides could decrease ROS and MDA levels while enhancing SOD activity [D. indusiatapolysaccharides. Readers should note that the skn-1 gene in C. elegans encodes a transcription factor that resembles the mammalian nuclear factor erythroid 2-related factor 2 (Nrf2). On the other hand, the DAF-16/FOXO transcription in C. elegans is the mammalian equivalent of the Forkhead transcription factors with the DNA binding domain or Forkhead box (FOX). This transcription factor is involved in diverse cellular function including the regulation cell death or apoptosis, resistance to oxidative stress and increased life span. Good references for the regulation of these transcription factors are available [C. elgans are activated by D. indusiata polysaccharides. In agreement with this data, the acidic and alkali extracted polysaccharides have been shown to increase SOD and GPx activities in the d-galactose-induced senescence in rats [The antioxidant activities of polysaccharides have also been demonstrated in animal models. Under parquet-induced oxidative conditions in activity . It alsovailable ,55,56. H in rats .β-glucans [D. indusiata polysaccharides in pathologies associated with ROS and/or oxidative stress.In high fat-induced oxidative damage model, the water soluble polysaccharides administered in mice could abolish the increased LPO (MDA level) while increasing the antioxidant status by elevating SOD, GPx, CAT and total antioxidant capacity (T-AOC) contents/activities in the liver and kidney tissues ,35. Simi-glucans . More im-glucans . All theC. elegans models, Zhang et al. [D. indusiata polysaccharides under various conditions. In addition to the above-mentioned antioxidant effects , for example, did not display cytotoxicity against mouse sarcoma S180 cells in vitro when tested up to 1 mg/mL though it displayed impressive effect in vivo . In contTo enhance the cytotoxic activity of the purified polysaccharide, DP1, Liao et al.  also preD. indusiata polysaccharides. In S180 tumor bearing mice, the triple helical polysaccharide (PD3) administered intraperitoneally (i.p.) for ten days has been shown to suppress tumor size and reverse body weight loss [β-(1→3)-D-glucans and α-(1→3) linked D-mannan polysaccharides from D. indusiata have also been shown to suppress tumor growth in S180 tumor bearing mice [Several animal experiments have also been employed to demonstrate the potential anticancer effect of ght loss . This coing mice . The maxD. indusiata compounds described in the following sections under these two contrasting conditions should not therefore be seen as a contradiction.Readers should make the distinction between therapeutic application via an immunestimulatory effect and immunosuppression. Polysaccharides could do both, and while immunostimulation is applicable when the immune system is suppressed such under cancer pathology, immunosuppression is applicable under chronic inflammatory conditions such as colitis, sepsis, etc. The effect of D. indusiata polysaccharides has been demonstrated in unstimulated macrophages in vitro. Treatment of the murine RAW264.7 cells with these polysaccharides could induce a proliferative response while markers of macrophage activation such as cytokines , NO synthase (level of NO) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) p65 were upregulated [β and TNF-α) expression, and NF-κB activation and nuclear translocation, Dectin-1 has been shown to induce the phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2), JNK1/2 and p38 mitogen-activated protein kinase (MAPK) [Laminaria japonica is through TLR4 as a recognition receptor leading to ERK1/2, JNK1/2 and P38 activation, NF-κB p65 mobilization from the cytoplasm to nucleus, and cytokines and NO overproduction in macrophages [The immunestimulatory effect of egulated . Moreoveegulated . In addie (MAPK) . As parte (MAPK) ). It hasrophages .β-(1→3)-glucan with side branches of β-(1→6)-glucosyl unit had a triple-helical structure. It promoted the proliferation of RAW 264.7 macrophage along with induction of NO and cytokine  production. Animal experiments using the acid and alkali extractible polysaccharides also showed increased macrophage phagocytosis power and NK cells killing activity  [The study by Fu et al.  on immunextract) .D. indusiata polysaccharides to cancer therapy was highlighted by the study of Han et al. [α.A classic example of immnunostimulation and a further potential application of n et al.  which emn et al. . The tumD. indusiata polysaccharides. At fairly small doses (25–50 μg/mL), the LPS-induced cytokine (IL-1) and ROS production, as well as TLR4 expression and NF-κB activation/nuclear translocation, were suppressed. By using the carrageenan-induced oedema model of the classical inflammation model and scalded edematous hyperalgesia in rats’ hind paws, Hara et al. [D. indusiata β-glucans.Wang et al.  employeda et al.  were theD. indusiata β-glucans was based on the dextra sulphate sodium (DSS)-induced colitis model in mice [α, IL-6, IL-1β and IL-18 and myeloperoxidase (MPO) activity. At the biochemical level, apoptosis markers were suppressed, tight junction proteins (TJP-1 protein expression) were abundant, the expressions for nucleotide-binding domain leucine-rich repeats family protein 3 (NLRP3), phosphorylated signal transducer and activator of transcription 3 ((p)-STAT3) and p-IκBα  enhanced, and M1 macrophage (F4/80+CD11b+ cells) polarization were down-regulated while the M2 (F4/80+CD206+ cells) subsets in splenic tissues were raised. Another study using the DSS-induced colitis model in mice was that employed by Kanwal et al. [One of the best anti-inflammatory activity studies for the  in mice . In thisl et al.  where thD. indusiata polysaccharides in the ulcerative colitis model demonstrated by Wang et al. [Firmicutes and increased Proteobacteria levels under a similar DSS-induced colitis model in mice were reversed by the administration of the crude polysaccharides [D. indusiata polysaccharides on gut microbiota came from the study by Kanwal et al. [D. indusiata polysaccharides have been shown to recover the altered  gut microbiota composition. More specifically, they have reported that the polysaccharides enhance the level of beneficial flora, such as Lactobacillaceae (lactic acid-producing bacteria), and Ruminococaceae (butyrate-producing bacteria). Moreover, the lipopolysaccharides were shown to suppress endotoxemia and the level of pro-inflammatory cytokine TNF-α, IL-6, and IL-1β, while the expression of tight-junction associated proteins  were increased. All these anti-inflammatory effects coupled with improvement of the gut microbiota and restoration of tight junction and mucus barriers demonstrated by Kanwal et al. [D. indusiata polysaccharides.In addition to the above-mentioned anti-inflammatory properties of g et al. , the deccharides . Anotherl et al. . Under bl et al.  imply thD. indusiata were administered in mice orally for 45 days. As shown in D. indusiata polysaccharides obtained by enzyme assisted or alkali extraction method , its biosynthesis pathway and activities have been the subject of many studies [D. indusiata can inhibit the growth of bacteria and fungi. The reported activity was however not of therapeutic relevance and was 200 mg/mL. Since the known antibiotic principle is a lipophilic terpenoid, albaflavenone, the poor antimicrobial activity of the water extract only suggests the lack of water soluble antibiotic agent in the fungi.Chen et al.  describe studies ,62,63,64 studies  also repD. indusiata has been shown to display tyrosinase inhibitory effect in a non-competitive manner [50, 0.35 mM) but not 2-furoic acid was active suggesting the role of the aldehyde functional group for the observed activity. On the other hand, 5-methyl furfural  had comparable inhibitory effect as 5-(hydroxymethyl)-2-furfural . We should note however that this level of potency and the non-competitive nature of inhibition do not make 5-(hydroxymethyl)-2-furfural as a significant lead compound for the discovery of novel tyrosinase inhibitors as depigmentation agents in medicine/cosmetics or anti-browning agents in the food/agricultural industries.5-(hydroxymethyl)-2-furfural isolated from the methanolic extract of e manner . In viewe manner  also scrHohenbuehelia serotina have been shown to inhibit the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and inhibit leukemia (L1210) cells and lymphoma (MBL2) cells proliferation [Wang and Ng  isolatedferation .D. indusiata is a common food particularly in the far eastern countries such as China and Japan. Its toxicity is thus not of a concern unless excessive amounts of, for example, extracts, and isolated compounds are consumed. The water-soluble polysaccharides [D. indusiata.charides  and thoscharides  have beeAgaricus, Auricularia, Ganoderma, Lentinus, Phellinus, Pleurotus and Trametes genera have been reviewed [D. indusiata only begun in earnest during the 1980s. Interestingly, the major components and biologically active compounds in this fungus are the carbohydrate class like those considered as the bioactive principles in the genus Ganoderma and Trametes. The plethora of evidences presented in the scientific literature for polysaccharides of these higher basidiomycete fungi as antioxidant, organoprotective, anticancer, immunomodulatory, antidiabetic/antiobesity, etc. are thus in good agreement with the assertion of therapeutic potential outlined in this communication for D. indusiata. Although mushrooms contain a wide range of polysaccharides such as chitin, mannans, galactans, and xylans, the prevalent bioactive polysaccharides in the higher medicinal mushrooms is β-glucans of the β-(1→3) linkages with some β-(1→6) branches [. In this context, studies on the physicochemical characterization ofD. indusiata polysaccharides so far has revealed important structural information on the general structural backbones of the bioactive compounds although more work is needed to isolate and characterize all the individual polysaccharides. In comparison to the polysaccharides, studies on the small molecular weight bioactive compounds of D. indusiata is still in its infant stage. Although, some terpenoids and alkaloids have been characterized with limited pharmacological effects, the lipophilic or small molecular weight secondary metabolites that could be manipulated in culture by biotechnology tools remains to be investigated.In comparison to plants, mushrooms are the growing but yet underexploited natural resources both as food and as sources of valuable medicines. In recent years, considerable attention has been given to higher basidiomycete which are considered as medicinal mushrooms based on their nutritional value and therapeutic implications. The significance of bioactive molecules from such mushrooms like the reviewed . One advbranches ,69,70. ID. indusiata polysaccharides is moderate at best. In recent years however, compounds that are not optimized for direct ROS scavenging have been shown to display antioxidant effects in vivo owing to their ability to boost antioxidant defenses such as SOD, GPx, and CAT. In this connection, an emerging mechanism of action for such natural products is the redox sensitive transcription factor Nrf2. Hence, compounds with weak radical scavenging effects can induce considerable antioxidant effects in vivo by upregulating the expression of Nrf2 leading to the activation of antioxidant genes and proteins including glutamate-cysteine ligase, glutathione peroxidase 1, thioredoxin reductase 1, NAD(P)H-quinone oxidoreductase 1, glutathione-S-transferase, SOD, CAT, peroxiredoxin, ferritin and HO-1 [D. indusiata polysaccharides to increase this antioxidant status under various experimental conditions in vitro and in vivo suggest their wider potential as antioxidant agents. Since oxidative stress is associated with major pathologies such as cancer, inflammation, neurodegeneration, cardiovascular diseases, etc., the demonstration of potential application of D. indusiata polysaccharides in the various experimental models could also be an attribute of antioxidant mechanism of action.Although a direct radical or ROS scavenging effect is a function of the structurally optimized phenolic compounds such as gallates, phenyl propanoids and flavonoids, the antioxidant effects of polysaccharides appear to represent the most studied biological activity of medicinal mushrooms. As expected, the direct radical/ROS scavenging effect of and HO-1 ,72,73. Tα; enhancing macrophage polarization and activity, and direct anticancer effect in vitro and in vivo, D. indusiata polysaccharides appear to display potential as anticancer agents through two distinct mechanism of action: direct antitumor effect and immunostimulation. This is in line with other medicinal mushroom polysaccharides such as those from Coriolus versicoloris [D. indusiata polysaccharides is also in line with what has been established for C. versicolor polysaccharides [D. indusiata polysaccharides have been established in a variety of experimental conditions both in vitro and in vivo. Hence, under pathological conditions such as LPS-induced macrophage activation and endotoxemia and experimentally induced gut inflammation such as colitis and antibiotic damage or neurodegenerative models, the anti-inflammatory effect of these polysaccharides are evident.By boosting the level of expression of proinflammatory cytokines such as IL-2, IL-6, and TNF-icoloris ,75. The charides . On the D. indusiata appear to ameliorate intestinal damage and inflammation while increasing the proportion of the gut microbiota associated with good health  and reducing the composition of those linked to pathologies.In recent years, there has been a growing understanding that alteration in the composition of the gut microbiota is a major risk to the development of many diseases. Therapeutic approaches to normalize the Bacteroidetes and Firmicutes composition have thus been employed to improve food digestion/bioavailability, improve the immune response and protect the barrier function of the gut, and even to modify the metabolism of drugs and xenobiotics. The prebiotic nature of mushroom polysaccharides as a mechanism for their therapeutic effects and how they alter the intestinal microbiota composition have been extensively researched ,78. In lD. indusiata is just beginning to take shape and the available data so far is encouraging to do more compressive research in this field. The studies by Wang et al. [Ganoderma lucidum [G. lucidum have also been shown normalize [The antiobesity and antidiabetic potential study on g et al. ,35,48 weg et al. . A more  lucidum ,80,81. Aormalize ,83.D. indusiata extracts and compounds are increasing in recent years and show therapeutic potential for various pathologies. Since the mushroom is considered as non-toxic with a significant volume of consumption especially in the Far East, more comprehensive studies on the small molecular weight constituents and polysaccharides are well merited. One other area of interest is the diversification of research to maximize the accessibility of the bioactive compounds and their yield through biotechnology tools.Overall, studies both on the chemistry and pharmacology of


Background: Vitamin B12 (B12) deficiency and excess are associated with increased risk of age-related-diseases and mortality. It has been suggested that high- and low-B12 concentrations link to increased mortality through accelerated genomic aging and inflammation. Evidence to support this is limited.Results: B12 was associated with all-cause-mortality, RTL and hsCRP in a non-linear fashion. The association between B12 and mortality was not independent, as it lost significance after adjustment for potential confounders. In the lowest-(LB12) and highest-(HB12) quartiles of B12 mortality was higher than in the mid-range . We divided subjects with LB12 in quartiles of their RTL. Those with the longest-telomeres had a lower mortality-rate  and lower homocysteine than those with the shortest-telomeres. Amongst subjects with HB12, those with long-telomeres also had a lower mortality than those with short-telomeres . IL-6 and hsCRP concentrations were low in HB12LT but were high in HB12ST.Methods: B12, homocysteine, telomere length (RTL), interleukin-6 (IL-6) and high-sensitive-C-reactive-protein (hsCRP) were measured in 2970 participants of the LURIC study.Conclusions: Mortality, stratified according to B12 and RTL, seems to be driven by different mechanisms. In LB12 and HB12 subjects, mortality and accelerated telomere shortening might be driven by homocysteine and inflammation, respectively. Short telomeres –3 and hyHHCY, induced by B12 deficiency, has been linked to mortality in several studies , 14–16. th quartile) had a markedly lower hazard ratio (HR) for all-cause (0.42) and CVD (0.40) mortality than those with the shortest telomeres (1st quartile) [Increased mortality has also been reported in subjects with telomere dysfunction –3. TelomUntil today, the relationship between telomere length and B12 has not been studied comprehensively. The very few existing studies reported no significant association –24. Howest quartile, mid-range and 4th quartile, n=3312). During a median follow-up period of 9.9 years 2321 patients stayed alive and 995 died. Of the 2970 participants in whom the measurements of B12 and RTL were complete, 2067 patients stayed alive and 903 died.The general characteristics of the study population were previously reported . Brieflyst and 4th quartile of B12  quartile of B12 were characterized by a 12.3 % higher risk to die during follow-up compared to those in the mid-range of B12    Model 1. n=1650)  Model 2. n=1650)  Model 3, n=1650)  Model 4.th  quartile of B12 plasma concentration were characterized by a 12.4 % higher risk to die during follow-up compared to those in the mid-range of B12  (Subjects in the 4 n=1650)  Model 1. n=1650)  Model 2, n=1650)  Model 3, n=1650)  Model 4.st and 4th quartile of B12 with higher mortality, we analyzed age-corrected RTL, another import biomarker of all-cause mortality [To further analyze the association of subjects in the 1ortality . The stast age-corrected RTL quartile  subjects had a lower mortality-rate compared to all the other subjects . The CoxFurther, the LB12LT (n=174) subjects were characterized by significantly lower concentrations of HCY compared to all the other subjects .HB12LT subjects  were characterized by a significantly lower mortality-rate compared to all the others . In addind and 3rd quartile) of B12 and are reported in Finally, the HB12LT subjects (n=203) were characterized by significantly lower concentrations of markers of inflammation like IL-6 and hsCRP . The Coxst and 4th quartiles of B12 and with age-corrected RTL in the lower three quartiles had higher mortality rate , compared to those in the 4th quartile of age-corrected RTL.Finally, subjects in the 1st quartile of B12 concentration compared to those in the mid-range, although the difference was not statistically significant  have a lower average HCY concentration and a reduced mortality compared to those with the shortest telomeres (LB12ST). Both, HHCY [Here we show that patients in the 1th, HHCY , 15, 16 th, HHCY  are estath, HHCY . TherefoIn addition to the putative genomic effects of HCY, there are several other aspects that may contribute to cardiovascular mortality and the occurrence of cardiac arrhythmias , 33. Forst quartile of B12, but also in the 4th quartile. Although high B12 has been linked to an increased mortality in previous studies [The present results show an increased mortality not only in the 1 studies –20 this  studies . In the  studies . In anot studies . A largeAt this point it should also be mentioned that not all previous studies were able to show an increased mortality in individuals with high B12 concentrations –40. DiffAnother important observation in the present study was that high B12 also lost its predictive power after adjustment for RTL and inflammatory markers. Patients with high B12 and long telomeres (HB12LT) were characterized by a lower mortality rate and by lower concentrations of hsCRP and IL-6. Based on these results it can be speculated that in subjects with high B12 concentrations systemic inflammation increases mortality risk through accelerated telomere shortening. This concept is supported by a previous study in which we showed a negative correlation between RTL and markers of inflammation . Other sst and 4th quartile of B12, respectively, mortality was highest in those with the shortest telomeres. In a previous analysis of the LURIC study we could show that participants with the longest telomeres had a 42% lower risk to die compared to those with shorter telomeres [Only few studies analyzed the relationship between B12 and RTL –24. Noneelomeres . As mentelomeres , 47, 48.st and 4th quartile of B12? It can be speculated that accelerated telomere shortening in B12 deficient subjects might be counteracted by compensatory mechanism that activate telomerase, an enzyme that can prolong telomeres. In oncologic patients, the induction of telomerase is a common tumor escape mechanism [Another open question is why average RTL is longer amongst individuals within the 1echanism . In contechanism  it can bAs B12 is an essential nutrient information about diet, calorie intake and vitamin supplementation could be of interest in this study. Only 81 subjects of the 3316 participants of the LURIC study were supplemented with vitamins. Because of such a small number we did not consider this group separately for statistical evaluation. Furthermore, detailed dietary information was not available.It should be mentioned that total B12 is not the best surrogate marker for assessing the B12 status , 27. TheIt also has to be acknowledged that extrapolating the impact of a single factor, such as B12, in a heterogeneous cohort of patients in which some have received polytherapy harbors substantial limitations that needs to be considered when interpreting the present results.Finally, this examination has been conducted in Caucasian persons scheduled for coronary angiography who are at intermediate to high risk of dying. Our conclusions therefore must not unconditionally be extrapolated to other ethnicities of persons with different baseline characteristics.Extreme B12 concentrations are associated with increased mortality risk. Amongst individuals with low or high B12 those with the shortest telomeres have the highest risk to die. Accelerated telomere shortening might be a common promotor of mortality in these individuals. Our results further suggest that B12 and RTL are differentially regulated, and the mechanisms causing short telomeres in subjects with extreme B12 concentrations are not the same. While HHCY might drive telomere shortening in the context of B12 deficiency, systemic inflammation is a potential accelerator of telomere attrition in individuals with high B12. In view of the limitations associated with the measurement of B12 and RTL, additional markers, such as HCY, IL6 or hsCRP, should be considered when assessing patient’s mortality risk.We analyzed baseline blood samples and clinical outcome data from 2970 Caucasian participants of the LURIC study (n=3316) in whom the measurements of B12 and RTL were complete. A detailed description of the LURIC study has been published . BrieflyMedian follow-up time was 9.9 years. Information about survival was obtained from local person registries. Two physicians blinded to participant’s baseline characteristics classified causes of death by reviewing hospital records and death certificates. In the case of disagreement about classification, the final decision was made by one of the principal investigators of LURIC study (W.M) after appropriate review of the data. The study was approved by the ethics committee of the Physicians Chamber of Rheinland-Pfalz and performed in accordance with the declaration of Helsinki . All par6 (n=3311) was measured by HPLC .B12 (n=3312) and folate (n=3315) were measured with automated immunoassays on an AXSYM analyzer  , high-se-ΔΔCt) [RTL (n=2974) was measured in genomic DNA using a quantitative-polymerase chain reaction (Q-PCR)-based assay, as previously reported . Briefly-ΔΔCt) . This me-ΔΔCt) .th-90th percentiles) for normally and non-normally distributed variables, respectively. Where indicated, quartiles of the entire study cohort were used. We generated quartiles of B12 and age-corrected RTL and the following four groups: LB12LT , HB12LT , LB12ST , and HB12ST .All data were examined for normality of their frequency distribution using the Kolmogorov-Smirnov test. Non-normally distributed variables were log-transformed prior to further statistical testing. Descriptive statistics provide means (±SD) or medians (10http://www.r-project.org). Kaplan-Meier plots were drawn using the R-package ‘survminer’ (v5.1-1).The Kruskal-Wallis test was used to identify differences between multiple groups of continuous variables. The Mann-Whitney-U test was used to compare continuous variables between two independent groups. The chi-square test was used for categorical outcomes. Multiple regression analysis was performed using a backward variable selection. Correlation analyses were performed according to Pearson. The Cox proportional hazard model was used to examine the association between quartiles of B12 and time to death from any cause. Kaplan-Meier curves were calculated to evaluate the cumulative survival during follow-up, according to quartiles of B12 and age-corrected RTL. All tests used were 2-sided and p values <0.05 were considered statistically significant. All statistical analyses were performed using SPSS , and R v3.4.1 (


Botryobasidium botryosum genome mining. FIP-bbo has the typical characteristics of FIP but is genetically distant from other FIPs. Recombinant FIP-bbo (rFIP-bbo) was produced in an optimized E. coli expression system, and the pure protein was isolated using a Ni-NTA column. Antineoplastic experiments suggested that FIP-bbo is similar to LZ-8 in inhibiting various cancer cells  at lower concentrations, but it is not as potent as LZ-8. The molecular mechanism by which FIP-bbo, FIP-fve, and LZ-8 are cytotoxic to cancer cells has been discussed based on molecular dynamics simulation. Point mutations that may improve the thermal stability of FIP-fve and FIP-bbo were predicted. These results not only present a new candidate protein for the development of anticancer adjuvants, but also provide an approach for designing FIPs with high anticancer activity.Fungal immunoregulatory proteins (FIP) are effective small molecule proteins with broad-spectrum immunomodulatory and anti-cancer activities and can be potential agents for the development of clinical drugs and health food additives. In this study, a new member of FIP named FIP-bbo was obtained through  FIPs possess a certain degree of homology with the heavy chain variable region of human and mouse immunoglobulins. Thus, FIP may be related to the immunoglobulin superfamily1. A comparison of the crystal structures of FIP isolated from Flammulina velutipes (PDB ID:1OSY)2, Ganoderma lucidum (PDB ID:3F3H)3, and G. microsporum (PDB ID:3KCW) showed that they are non-covalently linked homo-dimers, and each monomer comprises an N-terminal α-helix followed by a FNIII fold.Fungal immunoregulatory proteins (FIP) are small molecules obtained from various fungi that have similar amino acid compositions and protein structures4. The ability of these cytokines to produce a tumour suppressing environment indirectly proves that FIP regulates the immune system and has a cytotoxic effect on tumour cells5. Moreover, evidence show that FIP can directly inhibit cancer proliferation and migration by inducing apoptosis in tumour cells6.FIP have various immunomodulatory activities and are important to researchers. Several experiments showed that FIP is a potent T cell activator that enhances the immune response by modulating the production of important cytokines and molecular factors  in several vertebrate model systemsG. lucidum, inhibits A549 lung cancer cell progression by promoting EGFR degradation7. The FIP-fve, FIP from F. velutipes, inhibits A549 lung cancer cells migration and invasion by decreasing RacGAP1 expression and Rac1 activity8. Although both rLZ-8 and FIP-fve prevented lung cancer cell proliferation via the increase G1 arrest, their effect strengths are different. The anti-cancer effect of LZ-8 is stronger than that of the FIP-fve10.Despite being highly conserved with respect to its sequence and structure, FIP from different sources may possess different activity of fighting against cancer. Previous studies demonstrated that recombinant LZ-8 (rLZ-8), FIP from 11, FIP-ppl12, FIP-sch213, FIP-Lrh14, and FIP-dsq215 can be obtained by analysing the enormous genomic information available online.Only a few fungi contain FIP and considering the drawbacks related to fungal samples, it takes a lot of effort to identify new FIPs from the fruiting body or mycelium through isolation or cloning using a universal primer. However, the progress and affordability of genomic sequencing technology is transforming the way FIPs are discovered. Even without fungal samples, the gene sequence of FIPs such as FIP-nhaBotryobasidium botryosum, was obtained by performing a sequence similarity search in a rapidly evolving genome database. FIP-bbo is similar to LZ-8 in that it inhibits cancer cells  at lower concentrations; however, its activity was not as high as LZ-8. The FIP-bbo molecular model was constructed by homology modelling using LZ-8, GIM, and FIP-fve crystal structures as templates. The molecular mechanism by which FIP-bbo, FIP-fve, and LZ-8 induce cytotoxicity in cancer cells has been discussed using molecular dynamics simulation.Here, FIP-bbo, a novel FIP from The 336 base pairs (bp) FIP-bbo comprises 112 amino acids and has a molecular weight of 12.42 kDa. FIP-bbo with a pI value of 6.54 is an alkaline protein with 11 strongly basic (+) amino acids  and 11 strongly acidic (−) amino acids . FIP-bbo is a stable protein with an instability index (II) of 6.29 and lacks a signal peptide and transmembrane helices. As described above, FIP-bbo possess the typical characteristics of FIP.Ganoderma spp. , additionally, FIP-cru, FIP-tve, FIP-fve, FIP-nha, FIP-sch2, and FIP-sch3 were derived from the same evolutionary process. The FIP from Volvaria volvacea  and FIP-dsq2 were clustered into the second lineage. FIP-ppl and FIP-bbo alone formed a separate lineage. Sequence alignment analysis indicated that the central region is more conserved than the N-terminal and C-terminal region.The dendrogram based on the FIP amino acids analysis showed that FIP is mainly divided into three branches Fig. . The FIPE. coli. The expression conditions (induction temperature and concentration of the inducer) were optimized  in vitro by rLZ-8, rFIP-fve and rFIP-bbo was detected by flow cytometry (FCM)  had no remarkable inducing effect on apoptosis of Hela, Spca-1, and A549 cells  is considered to be an indicator of structural stability. RMSD analysis showed that each FIP monomer simulation system converges after 7 ns Fig. , LZ-8 di16. Most parts of the FIP structure were very stable (blue), and a few parts were flexible   and in FIP-fve  would increase the stability of FIP-bbo and FIP-fve. As shown as Fig. Temperature factor, also known as B-factor, is an ambiguity used to describe the state of atomic conformation in a structure. It reflects the conformational state of protein molecules in the structure. The higher the B-factor, the greater the ambiguity, and the more unstable or flexible the conformation of the corresponding partsed) Fig. . In the Through detailed structural analysis, 2001 protein conformations in the simulated trajectory of FIP are classified by the cluster analysis tool. This, combined with the results of protein docking analysis of the average protein conformation of each FIP cluster, showed that all protein conformations in the simulated trajectory were divided into two categories, one was the closed conformation, which could not form a homo-dimer similar to the crystal structure of FIP-fve/LZ-8, and the other was the opened conformation Fig. . In the On the basis of cluster analysis, the statistical proportion of the opened structure in the molecular dynamics trajectory is shown in Fig. In addition to the molecular dynamics simulation of FIP monomers, we also performed molecular dynamics simulation analysis of homo-dimers. And MM-PBSA calculations were applied for calculating binding free energies and its components of the FIP homo-dimer. The calculated results are listed in Table 17. FIP can be orally consumed and have high stability when exposed to denaturing conditions19. These characteristics make FIP a potential candidate to be used as an additive in the health food industry. In this study, a novel FIP, FIP-bbo, with anticancer activity was found from B. botryosum through genome mining. B. botryosum is a basidiomycete species that produces thin, resupinate, greyish to yellowish basidiocarps on wood debris and characteristic navicular basidiospores. But the ecology and nutritional strategy of this species, as well as other Botryobasidium spp., is not well known. Only some species have been collected from brown-rotted wood20. Therefore, to produce sufficient quantities of FIP-bbo for our analysis, a recombinant expression strain was developed.Numerous studies have shown that FIP possess broad spectrum immunomodulatory and anticancer activities, suggesting that they may be promising candidates in clinical disease therapies or for treatment as adjuvant agentsExperiments on cell proliferation, apoptosis and migration in cancer cells such as Hela, Spca-1 and A549 revealed that LZ-8, FIP-fve and FIP-bbo have anticancer activities, but their effective concentrations are different. At lower concentrations, both LZ-8 and FIP-bbo have potency in inhibiting cancer cells, but LZ-8 is more potent. FIP-fve has anticancer activities at higher concentrations, which is consistent with previous studies. These differences are closely related to the high-level structure of the FIP protein.23. Thus, this experiment used molecular dynamics simulation to study the dynamic changes of FIP in solution to explore and understand the molecular model of its anticancer activity. Through monomer and dimer simulations, the results obtained from stability experiments showed that LZ-8 was most stable, followed by FIP-fve and FIP-bbo . FIP-fve was in this state for a very short time (3.9% of the entire simulation), which was only one-tenth the amount of time that LZ-8 was in this state. This provides a theoretical basis for FIP-fve to be active only at higher concentrations. Increasing its concentration could increase the probability of forming a homo-dimer. On the contrary, LZ-8 has more time to form a homodimer, so LZ-8 can produce activity at lower concentrations. The time with respect to FIP-bbo (13.7% of the entire simulation) is lesser than LZ-8 but greater than FIP-fve. Therefore, the antitumor activity of FIP-bbo is similar to, but slightly lower than that of LZ-8. Although FIP-fve is less likely to form homodimers, it is very stable once dimers are formed. This may be the reason why the crystal structure of FIP-fve is a homodimer.B. botryosum. The physicochemical characterization of FIP-bbo is similar to the characteristics observed in the FIP. However, the FIP-bbo evolves independently in the two branches of FIP. Anti-proliferation, pro-apoptosis, and inhibiting migration experiments on Hela, Spca-1 and A549 showed that rFIP-bbo has anticancer activity. The anticancer activity of the rFIP-bbo lies between that of rLZ-8 and rFIP-fve. Molecular dynamics simulation analysis revealed the molecular mechanism observed of the anticancer activity shown by LZ-8, FIP-fve, and FIP-bbo through thermodynamics and molecular structures. The point mutations  may have improved the thermal stability. The study not only identifies a novel candidate protein for the development of an anticancer adjuvant, but also provides the basis for designing an FIP with high anticancer activity.FIP-bbo is a novel member of the FIP family mined from B. botryosum (GenBank: GCA_000697705.1) and amino acid sequences of LZ-8 (GenBank: ACD44335.1) and FIP-fve (GenBank: P80412.1) were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). The protein structures of LZ-8 (PDBID: 3F3H) and FIP-fve (PDBID: 1OSY) were obtained from the Protein Data Bank (PDB). A BLAST search was performed using the BLAST software. The MEGA7 package24 was used for reconstruction of a phylogenetic tree, and neighbour-joining trees were inferred by the program.The genome sequence of 25, homology modelling of FIP-bbo was performed with the crystal structure of FIP using G. lucidum (PDBID: 3F3H), G. microsporum (PDB ID:3KCW), and F. velutipes (PDBID: 1OSY) as templates. Using MODELER, 50 models were produced and ranked by the value of the objective function and the score of discrete optimized potential energy. The model with the highest score was selected as the three-dimensional (3D) structure of FIP-bbo and optimized by a 1000-step energy minimization process using the steepest descent method. The quality of the FIP-bbo 3D structures was evaluated using PROCHECK26.Using a protein modelling software, MODELLER27, with CHARMM2728 force field parameters and periodic boundary conditions. The initial models were solvated in 7 × 7 × 7 nm boxes with SPC water and a space of 10 Å around the solute. Sodium and chloride ions were randomly placed to neutralize and maintain the systems’ ionic concentration of 153 mmol/L. Before molecular dynamics simulation, the minimization with the steepest descent algorithms of 50000 steeps and two phases equilibration (NVT ensemble and NPT ensemble) was conducted. Finally, a 20 ns molecular dynamics simulation was performed at 310 K on the whole system. The time of the simulation was set at 2 fs, and the coordinates were documented for analysis every 1 ps. Post-processing the data was analysed using standard GROMACS tools and customized Perl scripts. The visualization of the refined model was performed by PyMOL software29.The monomers and dimers of LZ-8, FIP-fve and FIP-bbo were simulated for 20 nanoseconds (ns), respectively. The molecular dynamics simulations were performed using GROMACS v5.0.2 simulation package30 and RDOCK31. ZDOCK searches a space of exclusively symmetric multimers for the best structure using a grid-based Fast Fourier Transform (FFT) approach. Following which, the structural minimization and rescoring of initial-stage docking models was performed by RDOCK.The docking between the homo-monomers of LZ-8, FIP-bbo and FIP-fve were performed by ZDOCK34. The MMPBSA method for calculation of the binding energy in general can be summarized as:To study the interaction between the FIP monomers in the FIP dimer, the molecular mechanics Poisson Boltzmann surface area (MM/PBSA) analysis was performed using the g_mmpbsa tool of GROMACSdimer, Gmonomer1 and Gmonomer2 are the free energies of FIP dimer and FIP monomers in solvent, respectively. The free energies were estimated by:The GMM stands for the gas phrase energy, which consists of the electrostatic energy (Eele), the van der Waals interaction energy (Evdw) and the sum of the internal interaction (Eint). The solvation free energy (Gsol) is made up of the electrostatic solvation free energy (Gpolar) and the aplolar salvation free energy (Gnonpolar). Poisson–Boltzmann approaches can be used to calculate Gpolar and Gnonpolar is estimated from the solvent-accessible surface area (SASA).EE. coli and were synthesized by General Biosystems Limited. The expression vector used was PCreat-SII  to produce the recombinant FIP , and BamHI and XHoI were the enzyme cleavage sites. The recombinant plasmid was transformed into E. coli BL21 (DE3), and positive colonies were isolated. Its expression was induced by isopropyl-β-D-thiogalactoside (IPTG) and was visualized using SDS-PAGE. The His-tag and lipopolysaccharides (LPS) were removed.The genes encoding FIP-bbo (KDQ10166), LZ-8 (P14945), and FIP-fve (P80412) were optimized for 2 in an incubator. The Hela and A549 cells were resuspended to a concentration of 1–5 × 104 cell/mL in Dulbecco’s modified Eagle’s medium . The Spca-1 cell line were maintained in Roswell Park Memorial Institute 1640 medium  supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The effects on growth inhibition were evaluated by MTT method. Aliquots of 100 μL cell suspension and 100 μL rLZ-8/rFIP-fve/rFIP-bbo  were seeded into a 96-well microplate. PBS was used as negative control. After cultivation for 24 h at 37 °C in a humidified 5% CO2 incubator, the percentage of viable cells was measured by MTT assay. Absorbance (OD) was measured at 570 nm using a Multiskan MK3.Human cancer cell lines including cervical carcinoma cell Hela, lung adenocarcinoma cells A549, and Spca-1 were obtained from the Shanghai Institute of Biochemistry and Cell Biology and cultured at 37 °C under 5% CO6 cells/mL) and 200 μL rLZ-8/rFIP-fve/rFIP-bbo (8 μg/ml) in PBS were incubated in a 24-well microplate at 37 °C 5% CO2 for 24 h. Untreated cells were used as negative controls. The percentage of apoptosis was evaluated using the Annexin V-EGFP/PI Cell Apoptosis Detection Kit  and then analysed using a flow cytometer  with Cell-Quest software.To observe if rLZ-8/rFIP-fve/rFIP-bbo induced apoptosis, 800 mL tumour cell suspension , according to the manufacturer’s protocol. Staining was analysed using laser confocal fluorescence microscopy . The culture medium was used as a negative control.When cells die, chromatin contracted. Cover glasses were put in 6-well plates. Cells were inoculated into the plates at 5 × 10The wound-healing assay was used to compare the inhibitory effect of rLZ-8, rFIP-fve, and rFIP-bbo on the migration of the cells. The cells were cultured on 6-well plates and grown in a medium containing 10% FBS to a nearly confluent cell monolayer. A plastic pipette tip was used to draw a linear “wound” on the cell monolayer of each well. The monolayer was subsequently washed twice using PBS to remove debris or detached cells, and 8 μg/mL of rLZ-8, rFIP-fve and rFIP-bbo was added to the plates, respectively. PBS was added to the negative control. After 24 h and 36 h of incubation, the morphology of the cells was observed using an inverted microscope , and captured by a digital camera coupled to the microscope .All values presented are mean ± standard deviation (SD). Each experiment was repeated thrice. Statistical comparisons between treatments were calculated by unpaired t-tests using GraphPad Prism 5 Software . The values were significant when the p value was smaller than 0.05 between the two treatments.Supplementary Information


The investigation highlighted that the DART-MS method (~1.3 min per three samples) could be used as a high-throughput alternative to the classic UHPLC-ESI-QTOF method (~15 min per three samples).The routine spermidine (SPD) detection method is time-consuming and laborious due to the lengthy chromatographic separation and/or tedious sample derivatization pretreatment. In this study, direct analysis in real-time ionization mode coupled with mass spectrometry (DART-MS) was developed to rapidly determine the SPD content of 12 bean cultivars. The results were compared in detail with those of the classical UHPLC-ESI-QTOF method. After conducting a series of optimizations, a simple sample extraction procedure employing 80% aqueous methanol, was followed by determination of sample extracts directly without any chromatographic separation or prior derivatization. The validated method showed excellent performance with low limits of detection (LOD of 0.025 mg·kg Spermidine (SPD) is classified as a polyamine due to the number of amino groups in its chemical structure. It displays pleiotropic effects that include antioxidant properties and anti-inflammatory functions as well as improvement of mitochondrial metabolic function . In receBeans are widely consumed worldwide, especially in Asian countries. Bean seeds contain polyamines such as SPD, spermine, putrescine and cadaverine. In particular, the SPD level of the soybean is considerably higher than that of common cereals, vegetables, or animal food products . This miN-hydroxysuccinimidyl carbamate (AQC) [o-phthaldialdehyde [Various methods to measure the capsaicinoid content have been used by researchers. The determination of polyamines is performed by high-performance liquid chromatography (HPLC), gas chromatography, and capillary electrophoresis. The most common method is HPLC coupled to an ultraviolet detector (UVD) or fluorescence detector (FD). Due to the structure of SPD without any chromophore or fluorophore, the detection of SPD by UVD or FD requires complicated pretreatment steps with derivatization reagents such as 6-aminoquinolyl-te (AQC)  or o-phtaldehyde . Though Ambient mass spectrometry (AMS) refers to those techniques that have the capability of both sampling through desorption and ionization under open-air conditions for the subsequent MS analysis. Since first reported, over thirty different techniques have become available . AMS canm/z 146 along with several ion adducts in the background  ratio of DART-MS is higher than that of UHPLC-ESI-QTOF.An ultra-high performance liquid chromatography liquid chromatograph coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF) has been demonstrated to provide high separation capacity and accurate mass, which facilitates the elucidation of the fragmentation schemes of target compounds . To asseckground A. The pr-QTOF-MS B, the prm/z 75 and m/z 72 when the C–N bond of m/z 146 is broken; then, after the cleavage of the C–N bond from the m/z 146 species, the ions at m/z 75 and m/z 72 were obtained. The second cleavage pathway is m/z 146 undergoing an intramolecular deamination to form m/z 129 ion fragments, followed by loss of a neutral ammonia molecule to obtain m/z 112 fragment ions, and subsequent losses of C2H2 and CH3C≡CH to produce the fragment ions at m/z 84 and m/z 72, respectively. The third is the loss of a CH2=CH–CH=CH2 molecule from ion at m/z 112 to form an ion at m/z 58.The MS/MS spectra obtained by both these two methods are shown in m/z 146 was measured at different temperatures ranging from 250 °C to 500 °C. The relationship between the temperature and the response intensity of m/z 146 is shown in m/z 146 increased with increasing temperature near 450 °C, after which, the intensity started to decrease drastically beyond 450 °C. Therefore, 450 °C was selected as the gas heating temperature during the analysis.The gas temperature is an important ionization parameter in the analysis of SPD by DART-MS. The response intensity of SPD parent ion at + in the atmosphere and reducing the chemical background [m/z 146 was observed as the temperature was increased from 50 to 100 V, and the intensity was the highest at 100 V. The study by Harris also showed that the grid voltage may affect the sensitivity of the analysis [The grid voltage can perform a variety of functions, including acting as an ion repeller removing ions of opposite polarity, thus preventing the loss of signal caused by the recombination among of these ions . Furtherckground . As seenanalysis , and them/z 146 can be affected by the sliding speed of the sample glass. The response intensity of m/z 146 was measured and the results are shown in m/z 146 gradually decreases with increasing speed. Finally, the sample sliding speed is maintained at 0.6 mm/s. On the DART ion source, the sample was introduced into the mass spectrometer through a glass bar placed on a linear track. Therefore, the response intensity of m/z 72. In this study, the CE was optimized from 10 to 35 V  showed that the linear relationship is excellent. Although there is no chromatographic separation, the LOD of SPD (0.025 mg/kg) measured by DART-MS method was significantly lower than that of UHPLC-ESI-QTOF method (0.036 mg/kg). The intra-day precision of SPD was calculated by repeated determination of standard solution six times in the same day. The intra-day and inter-day precisions were 3.60% and 7.50%, respectively. While in the UHPLC-ESI-QTOF method, the intra-day and inter-day precisions were 2.15% and 5.30%, respectively. Therefore, the reproducibility of the DART-MS method is superior to the literature, which in soybeans ranged from 5 to 38% by HPLC-FD method [It is necessary to validate any newly developed analysis method. Both the DART-MS and UHPLC-ESI-QTOF methods exhibition good linearity, and the measured recoveries, LOD, LOQ, and precision are shown in D method . HoweverThe matrix effect generally occurs during the ionization of analyte, which can adversely affect the quantitative analysis of the target. In this experiment , the No.m/z 146 - 72) both in DART-MS/MS (+) versus the UHPLC-ESI-QTOF (+) method. As shown in The spermidine contents were quantified from the peak areas using the multiple reaction monitoring mode  was purchased from Sigma . Formic acid (HPLC grade) was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd.  SPD standards (≥98%) purchased from Shanghai Yuanye Biotechnology Co., Ltd. .v/v). All of standard solutions were stored at −18 °C before further analysis.Samples of beans (1.000 g) were weighed in a 50 mL centrifuge tube, 80% methanol (10 mL) was added and the mixture vortexed for 2 min. The mixtures were then sonicated in an ultrasonic bath for 20 min. After centrifugation of the extract, the supernatant was filtered and the extract was stored at −18 °C for further analysis. Working standard solutions of SPD ranging from 0.312 mg/L to 10 mg/L were prepared by diluting the SPD stock standard solution (20.0 mg/mL) with 80% menthol  connected to a 3500 triple quad mass spectrometer . SPD or sample solutions (2.0 µL) were deposited on the tips of glass sticks. These sticks were held on a linear rail, and then passed through the gap to introduce the sample ions into the mass spectrometer. The optimized DART settings were as follows: the distance between the DART ion source and the ion transfer tube was 1.0 cm; positive ion mode; gas temperature 450 °C; and grid electrode voltage 100 V. A constant speed of 0.6 mm/s was used for the Dip-It tip rail system. High-purity helium (99.999%) was used as the running gas.m/z 50–400 Da and the scan time is 0.1 s The mass condition was: ion source gas 1 and gas 2, 45 psi and 50 psi; curtain gas 35 psi; declustering potential 60 V; collision energy 25 V.A Nexera X2 UHPLC system  was employed for analyte separation. The system was equipped with a vacuum degasser, auto sampler, pump and PDA detector. The extracts were analyzed on a C18 column . Mobile phase A was acetonitrile, and mobile phase B was ultrapure water with 0.2% formic acid solution. According to a previously described method  and sligIn this study, the reliability of the method was verified by measuring spike recovery, mechanistic effects, limit of detection (LOD) , limit oThe standard curve of the matrix effect was made by serially diluting a series of standard solutions (10 mg/L) with the sample solution, and an equivalent concentration of the standard solution was diluted with 80% methanol as a blank. The calibration curve ranged from 0.312 mg/mL to 10 mg/mL. The matrix effect is calculated by the following formula ,23:(1)MaAll experiments were repeated six times. The results were mean ± standard deviation, and the significant difference was determined at 95% confidence interval by analysis of variance followed by Duncan’s least significant test. In summary, DART-MS was demonstrated to be a powerful analytical technique for the rapid quantitative analysis of SPD in beans. Good agreement between DART-MS and UHPLC-ESI-QTOF results was obtained for 12 bean samples, ng that the former could be used as a rapid alternative (~1.3 min per three samples) to conventional UHPLC-ESI-QTOF analyses (~15 min per three samples). This study indicated that DART-MS will greatly simplify the SPD analysis period due to the elimination of tedious derivatization steps and lengthy separation processes. The method is environmentally friendly due to the fact it does not use a toxic mobile phase. Furthermore, the results of this study showed that the bean varieties provide a good source of SPD.


In order to improve the interfacial properties of graphene oxide (GO) and epoxy resin (EP), hyperbranched polyesters with terminal carboxyl (HBP) non-covalently functionalized graphene oxide (HBP-GO) was achieved by strong π-π coupling between hyperbranched polyesters and GO nanosheets. The effects of non-covalent functionalization of GO on the dispersibility, wettability and interfacial properties were analyzed. The mechanical properties and enhancement mechanism of HBP-GO/EP composites were investigated. The results show that the hyperbranched polyesters is embedded in the GO layer due to its highly branched structure, which forms the steric hindrance effect between the GO nanosheets, effectively prevents the agglomeration of GO nanosheets, and significantly improved the dispersibility of GO. Simultaneously, the contact angle of HBP-GO with EP is reduced, the surface energy, interfacial energy and adhesion work are increased, then the wetting property of HBP-GO is significantly improved. The main toughening mechanism of HBP-GO is microcrack deflection induced by HBP-GO and plastic deformation of the EP matrix. In the microcrack propagation zones, HBP-GO may produce the pinning effect near the microcrack tips and change their stress state, resulting in microcrack deflection and bifurcation. So, the microcrack propagation path is more tortuous, which will consume much more fracture energy. Therefore, the mechanical properties of the HBP-GO/EP composites are greatly improved. Epoxy resin has been applied extensively in structural adhesives, electronic instruments, construction, coatings and other fields due to its high mechanical properties, good bonding properties, low cure shrinkage and good process performance. In particular, epoxy resin has become an irreplaceable and important material as a composite matrix ,2,3. HowThe functionalized treatment of graphene oxide (GO) opens up a wide range of application for resin-based composites ,16,17. TIn this study, hyperbranched polyester (HBP) non-covalently functionalized GO (HBP-GO) was prepared by using a relatively simple processing method, and the HBP-GO reinforced EP matrix composites were produced by solution casting. The infiltration property of HBP-GO was discussed by the test of contact angle, and the interface properties were analyzed by quantitative calculation of the interface energy and the adhesion work. The fracture morphologies of HBP-GO reinforced epoxy composites were observed, and the enhancement mechanism of HBP-GO was expounded, which provided a reliable basis for the application of GO-reinforced resin-based composites in engineering.N,N-Dimethylformamide (DMF), tetrahydrofuran (THF) and absolute ethanol were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China.Graphene oxide  used in this experiment was supplied by Nanjing JCNano Technology Co., Ltd., Nanjing, China. Hyperbranched polyesters (HBP-COOH) is supplied by WH Hyperbranched Resin Technology Co., Ltd., Wuhan, China. The epoxy resin (E51) was supplied by Wuxi Resin Factory Co., Ltd., Wuxi, China. The QS-1622 curing agent is provided by Beijing Qingda Qi Shi New Material Technology Co., Ltd., Beijing, China. (QS-1622 and E51 compound ratio is 1:2). First, 100 mg of HBP-COOH was dissolved in 20 mL of DMF. Then it was mixed with the DMF dispersion of GO and stirred with the ultrasonic dispersion at 24 °C for 1 h. Then the GO dispersion was allowed to stand for 1 h. Next, filtration was carried out using a polycarbonate (PC) with a diameter of 10 μm under the condition of low pressure and the functionalized GO was washed with excess absolute ethanol. The filtration and washing process were repeated 3 times, and the finally obtained product was dried in a vacuum oven until the weight is stable to obtain a hyperbranched polyester functionalized GO (HBP-GO).First, HBP-GO was dispersed in a THF solution and sonicated for 2 h. After sonication, the dispersion solution of HBP-GO/THF was prepared. Then, the dispersion solution of HBP-GO/THF was added into the EP. The mixture was sonicated at 60 °C for 2 h while the mixture was stirred at 2000 rpm using the high speed mechanical mixer. Finally, the mixture was degassed in vacuum for 24 h and then mixed with QS-1622 curing agent. After stirring for 10 min and evacuating at 70 °C for 1 h, the mixture was poured into the PTFE mold for curing. Moreover, in order to study the enhanced mechanism of functionalized graphene oxide reinforced EP (HBP-GO/EP), untreated graphene oxide enhanced EP (GO/EP) and pure EP samples were also prepared for comparison. The distribution is shown in −1, the scanning time is 32 times, and the scanning range is 500–4000 cm−1. The TGA was tested by a PL thermal analyzer . The test conditions were a nitrogen atmosphere with a heating rate of 20 °C/min. Scanning electron microscopy (SEM) measurements were carried out with a Hitachi S-4800 scanning electron microscope, city, country. Before this step, the tensile section of the GO/EP composite material needs to be sprayed with gold to enhance its electrical conductivity. The state of dispersion of GO and HBP-GO in the EP matrix was obtained by JEM-2100, JEOL transmission electron microscopy, city, country. The ultra-thin sections of the GO/EP composites sample with a size of 1 mm × 1 mm and a thickness of 50–60 nm was prepared by a diamond knife. The contact angle of the GO film with EP was performed by the optical meter OCA20, Dataphysics. The GO film was obtained by filtering under reduced pressure from a GO/THF dispersion having a content of 1 mg/ml. The filter membrane was polycarbonate (PC) with a diameter of 10 μm.The test result of FITR spectra were obtained using the Tensor 27 manufactured by Bruker Manufacturing, Germany. The scanning resolution is 4 cm3. The pre-cracking method of the sample is as follows: Firstly, a groove of 2 mm deep is processed by a small hand saw, and then the pre-formed crack is introduced into the bottom of the groove of the rectangular parallelepiped sample by a sharp blade. The experimental temperature was room temperature and the experimental loading rate was 1 mm/min. furthermore, at least 5 samples should be tested for the same component. The formula of fracture toughness (ICK) and critical energy release rate (ICG) is as follows [G is the shape factor of the microcracks, B is the sample thickness, E is the sample elastic modulus, W is the sample width and S is the span. Both the EP and its composites have a Poisson’s ratio (v) of 0.35 [The Shore hardness was obtained by the D-type tester. The fracture properties of the epoxy composite were tested in accordance with ASTM D5045-99 using a rectangular parallelepiped three-point bending specimen (SENB) having a sample size of 50 × 10 × 5 mm follows :(1)KIC=G of 0.35 .−1 (O-H stretch of carboxylic acid and hydroxyl), 1715 cm−1 (C=O stretch) and at 1049 cm−1 (C-O-C stretch). The FTIR spectrum of HBP shows characteristic peaks of methylene stretch in the range of 2853–2955 cm−1. As well, there is the stretching peak of O-H in 2520–2640 cm−1 range and the stretching peak of C=O in carboxylic acid groups at 1639cm−1. In addition, the peak for C=C stretching of the benzene ring of HBP is observed in the range of 1495–1611 cm−1. As well, 1230 cm−1 is ascribed to the stretching characteristic peak of C-O-C. Compared with the peak of unmodified GO, the HBP-GO nanosheets showed obvious methylene characteristic peak at 2923 cm−1. Moreover, C-O-C stretching vibration peak appeared at 1230 cm−1, and the O-H stretch and C=O stretch were obviously enhanced. These new absorption peaks generated on the HBP-GO indicate that HBP has been adsorbed on GO, and the HBP-GO has been successfully prepared.The FT-IR spectra of GO, HBP and HBP-GO are shown in The TGA test results of GO, HBP and HBP-GO are shown in The morphologies of GO and HBP-GO were researched by TEM imaging, as shown in In order to further study the dispersion state of GO and HBP-GO in EP matrix, ultrathin sections of GO/EP and HBP-GO/EP composites were observed by TEM . Some daContact angle could characterize the wetting properties of GO and EP matrix. By measuring the contact angle of GO and EP before and after functionalization, the wettability can be visually indicated. The measurement results are shown in θ), adhesion work, and surface energy can be obtained by the formula:Lγ, Sγ, and LSγ represent the surface energy of the liquid, solid, and the interface of solid-liquid. aW represents the adhesion work of the interface of solid-liquid. Based on Owens, D.K.J and Wendt, R.C.J [Spγ and Sdγ are the polar and non-polar components of solid surface energy, and Lpγ and Ldγ are the polar and non-polar components of liquid surface energy.In order to analyze the interface properties of HBP-GO, the interface energy and adhesion work were analyzed and calculated. According to the Young-Dupre equation , the cont, R.C.J , the intFrom Equations (3) and (5), Equation (8) is available:Lγ, Lpγ and Ldγ are selected to calculate the surface energy of GO. The solvent used in the experiment was glycerol and water with a large difference in surface energy components. The Lpγ and Ldγ of glycerol were 21.7 mJ/m2 and 41.9 mJ/m2, respectively, and the Lpγ and Ldγ of water were 52.1 mJ/m2 and 20.8 mJ/m2, respectively. Moreover, according to previous studies, the surface energy of EP is 41.5 mJ/m2 [According to the Equation (8), two kinds of solvents known as .5 mJ/m2 . The cal2. After functionalization with HBP, the surface energy of HBP-GO was significantly improved, which was 23.2% higher than that of original GO. By comparing the surface energy components of GO and HBP-GO, we can know that the increase of surface energy for HBP-GO is mainly because the improvement of non-polar components. The non-polar component of surface energy is caused by the interaction between the electron dipole and the induced dipole in the neighboring atom or molecule [2). After functionalization with HBP, the non-polar component of HBP-GO surface energy increases to 49.98 mJ/m2 due to the strong π-π interaction, which also gives HBP-GO a larger surface energy (59.65 mJ/m2). Meanwhile, the non-polar component of the surface energy reflects the non-polar part of the van der Waals force, which can characterize the morphology of the solid surface. Therefore, the increase of the non-polar component of HBP-GO surface energy indicates that HBP functionalized GO increases the GO surface roughness, which is consistent with the above-mentioned research results .Compared the interface energy and the adhesion work of the GO before and after functionalization , it can The test results of tensile properties of different content of GO nanosheets reinforced epoxy composites are shown in W), length (L), and thickness (t). The Halpin-Tsai equation of randomly oriented fibers is applied as follows [CE and ME represent the modulus of composites and the pure EP; α represents the ratio of CE to ME. GOV is the volume fraction of GO. The GO volume fraction is converted to a mass fraction according to the standard density of GO (F = 2.25 g/cm3) and the matrix density (F = 1.12 g/cm3) listed by the manufacturer. β and ζ are constants:In order to predict the elastic modulus of the epoxy composite, we assume that the GO nanosheet is a rectangular cross-section fiber with width –(13) into Equation (9), α can be calculated by the following formula:There is a reasonable agreement between the predicted and experimental values until 0.5 phr of GO for the composites. When it is bigger than 0.5 phr, the experimental modulus begins to fall below the prediction. As is shown in ICK and ICG of GO and HBP-GO reinforced epoxy resin composites. It can be found that the ICK and ICG of the pure EP are 0.75 MPa·m1/2 and 0.19 KJ/m2, respectively. The ICK and ICG of GO/EP and HBP-GO/EP are higher than those of pure EP and the HBP-GO has a better enhancement effect. When the HBP-GO content is 0.5 phr, the ICK and ICG of the HBP-GO/EP composites reach 1.01 MPa·m1/2 and 0.26 kJ/m2, which are increased by 35.7% and 36.8% than the pure EP and increased by 7.45% and 13.04% than those of 0.5 phr GO/EP (0.94 Mpa·m1/2 and 0.23 kJ/m2). The reasons are as follows: Firstly, This is because HBP-GO is dispersed in the EP matrix uniformly and the interfacial properties with the resin matrix is pretty good , the size of critical microcracks to different samples can be calculated :(15)C=2KThe morphologies of the fracture surfaces of GO/EP and HBP-GO/EP are shown in The tensile fracture surface of the HBP-GO/EP composite is shown in ICK and ICG of 0.5 phr HBP-GO reinforced epoxy resin composites reached 77.8 MPa, 3658.34 MPa, 1.01 MPa·m1/2 and 0.26 kJ/m2, respectively. Compared with pure epoxy resin, they were increased by 41.4%, 49.9%, 35.7% and 36.8%, respectively.(1) HBP-GO was successfully prepared using hyperbranched polyesters (HBP) by a non-covalent functionalization method. the hyperbranched polyesters is embedded in the GO layer due to its highly branched structure, which forms the steric hindrance effect between the GO nanosheets, effectively prevents the agglomeration of GO nanosheets, and significantly improved the dispersibility of GO. Simultaneously, the contact angle of HBP-GO with EP is reduced, the surface energy, interfacial energy and adhesion work are increased, then the wetting property of HBP-GO is significantly improved. The tensile strength, elastic modulus, (2) The fracture surface analysis showed that HBP-GO was uniformly dispersed in the EP matrix and the fracture surface was rough. The toughening mechanism of HBP-GO is the deflection and bifurcation of crack induced by GO and plastic deformation of the matrix. In the microcrack propagation zones, HBP-GO may produce the pinning effect near the microcrack tips and change their stress state, resulting in microcrack deflection and bifurcation. So, the microcrack propagation path is more tortuous, which can consume much more fracture energy. Therefore, the mechanical properties of the HBP-GO/EP composites are greatly improved.


A methodology is reported for the preparation of 2-aryl-benzimidazole-3-oxide derivatives. By means of a one-pot two-step protocol, a library of 42 new compounds has been prepared. Reactions were performed in a total time of 40 min using microwave heating as a tool. A streamlined work-up process was also developed, allowing for facile isolation of the products. The methodology offers a more sustainable approach than previous routes, with only water and ethanol being used as solvents, and the products being isolated by means of a simple filtration without the need for any further purification. Nitrogen-containing heterocyclic compounds play a vital role in drug development because they can increase binding efficiency and solubility as well as facilitate the formation of salts. These properties improve oral absorption and bioavailability of the drug ,2. The bN-oxide derivatives have a unique functionality, due to the N-O moiety that can act effectively as a push-pull electron donor/acceptor group; the N+-O− group being strongly polarizable in both directions [N-oxides with unique and interesting chemical [N-oxides show anti-protozoa activity when tested against Trypanosoma cruzi and Trichomonas vaginalis [Benzimidazole rections . In addirections ,14. Theschemical  and biolchemical  propertiaginalis . DespiteN-oxides is usually carried on through base-mediated cyclization reactions of o-nitroanilines [NAr reaction between a suitably-functionalized haloarene and an amine. This is followed by a cyclization. Both steps employ solvents such as 1,4-dioxane and dimethylformamide (DMF), which are undesirable from a sustainability viewpoint [N-oxides for screening in a biological assay, we have turned our attention to developing an improved protocol for their preparation. Microwave heating has proven an excellent tool in this endeavor.The synthesis of benzimidazole anilines  instead anilines . One appanilines ,21,22. Iiewpoint ,26,27,28iewpoint . This prN-oxide derivatives by a one-pot two-step methodology facilitated by microwave heating.Since the first reports in 1986 ,31, micrNAr reaction between o-nitrochlorobenzene derivatives (1a-c) and a range of different aryl amines (2a-n)  .3, in good to excellent yield. Rather than isolate this intermediate, our objective was to convert the 2-nitroanilines directly to the corresponding benzimidazole N-oxides. We achieved this by adding a quantity of 0.5 M aqueous potassium carbonate K2CO3 and subjecting the reaction mixture to a further heating cycle of 20 min at 120 °C. After this time, the crude product mixture was assayed using 1H-NMR spectroscopy. In all cases, conversion to the desired benzimidazole N-oxide, 4, was greater than 90%, the remaining material being identified as unreacted intermediate 3.Optimal conditions were found to involve heating the reaction mixture for 20 min at 120 °C. This led to the formation of the desired aniline product, N-oxides in high purity. This proved to be the most challenging aspect of the study. We initially followed a literature protocol [3, since this is preferentially soluble in the organic layer. Acidification of the aqueous phase in order to protonate the benzimidazole derivative, followed by extraction with ethyl acetate, gave the desired 2-aryl-benzimidazole N-oxide derivative, 4, in pure form. We followed this work-up protocol for four representative examples, but in each case, the isolated yield obtained was unacceptably low  are soluble in the reaction medium, meaning that product isolation is challenging. Cooling the product mixture in the vessel using an ice bath led to further precipitation. Filtering the solid and allowing it to dry furnished us with the desired product in pure form. Not only does this approach allow us to obtain the products in far superior isolated yield as compared to the previous purification protocol  attached to the 2-position of the benzimidazole ring played a key role in the precipitation process. This made us probe the acid-base characteristics of benzimidazole ilibrium  and the terature ,22. The protocol , it alsoach case . The syn1d) as a substrate, but no benzimidazole N-oxide formation was observed. This follows the trend previously noted for similar substrates [3 reduces the acidity of the NH proton. The deprotonation of 3 is proposed as the first step of the cyclization mechanism, and thus, reducing the propensity for this impacts the efficacy of the reaction [In an attempt to broaden the substrate scope of the reaction, we first turned attention to the nitrochlorobenzene component. We attempted the reaction using 2-nitrochlorobenzene  were performed at 300 K using either a Brüker Avance Ultra Shield 300 MHz, Brüker DRX-400 400 MHz, or Brüker Avance 500 MHz spectrometer . 1H NMR spectra were referenced to residual non-deuterated dimethylsulfoxide (2.50 ppm) in deuterated dimethylsulfoxide (d6-DMSO). 13C NMR spectra were referenced to DMSO (39.52 ppm). 19F NMR spectra were referenced to hexafluorobenzene (−164.9 ppm). Melting points were determined with a Barnstead Electrotherm 1001D Mel-Temp capillary melting point apparatus in open capillaries and are uncorrected. High-resolution mass spectra were performed on a JEOL AccuTOF-DART SVP 100 unit employing a positive direct analysis in real time (DART) ionization method, using polyethylene glycol (PEG) as the internal standard. Reactions were monitored by 1H NMR spectroscopy.All reactions were performed using a CEM Discover SP monomode microwave unit, in a closed vessel. Temperature was measured by means of an infrared (IR) temperature sensor located in the floor of the microwave unit. Nuclear magnetic resonance (NMR) spectra (d6-DMSO) was purchased from Cambridge Isotope Laboratories. The o-nitro-chlorobenzene derivatives were purchased from Oakwood Chemicals or Alfa Aesar. Ethanol was obtained from Pharmaco. Potassium carbonate was purchased from J. T. Baker. Hexafluorobenzene was purchased from Oakwood Chemicals. All amines employed were purchased from Oakwood Chemicals and used without purification.Deuterated dimethylsulfoxide (4aa): 2-Chloro-1,3-dinitrobenzene , benzylamine  and ethanol (3 mL) were added to a 10 mL-capacity glass tube equipped with a magnetic stir bar. The reaction mixture was sealed with a septum and placed into a CEM Discover SP microwave unit. The contents of the vessel was heated to 120 °C using an initial microwave power of 100 W and held at this temperature for 20 min, the microwave power automatically fluctuating to hold the reaction mixture at the desired temperature. The reaction mixture was stirred constantly. After the allotted time, the reaction mixture was allowed to cool to below 50 °C before taking the vessel out of the microwave unit. The septum was removed, 0.5 M aqueous potassium carbonate (2 mL) was added, and the septum was then replaced. The mixture was then replaced into the microwave unit and heated at 120 °C for 20 min using an identical protocol to that for the first step of the reaction. After cooling, removing from the microwave unit, and de-capping, water (5 mL) was added directly into the glass reaction vessel. The product mixture was acidified with 2 M hydrochloric acid to a pH of between 2 and 5 at which point a yellow solid precipitated from the solution. The stir bar was removed and the reaction vessel placed into an ice bath for 1 h to promote further product precipitation. After this time, the solid product was removed by filtration and dried at 80 °C. Analytically pure 4aa was obtained as a yellow powder ; characterized by melting point determination, 1H- and 13C-NMR spectroscopy, and high-resolution mass spectrometry (see the 7-Nitro-2-phenyl-1H-benzimidazole 3-oxide ( see the . Using a one-pot-two step protocol, a library of 42 novel 2-aryl benzimidazole-3-oxide derivatives were prepared. The use of microwave heating as a tool greatly facilitated the procedure, a total reaction time of 40 min being all that was needed. Ethanol and water were employed as solvents. This, in conjunction with a work-up protocol that involves a simple precipitation and filtration, thereby obviating the need for aqueous-organic extraction, offers a sustainable route to the synthesis and isolation of this class of compound. The products obtained bear diverse structural characteristics, thereby furnishing them with potentially interesting biological activity; this is an area of ongoing study in our group. In addition, post-synthesis modification of the benzimidazole N-oxide products could further yield compounds of biological and chemical interest.